The phthalocyanine pigments are a group of light-fast organic pigments with four isoindole groups, (C.sub.6 H.sub.4)C.sub.2 N, linked by four nitrogen atoms to form a cyclic conjugated chain. Included are phthalocyanine (blue-green), copper phthalocyanine (blue), chlorinated copper phthalocyanine (green), and sulfonated copper phthalocyanine (green). These pigments are commonly used in enamels, plastics, linoleum, inks, wallpaper, fabrics, paper, and rubber goods.
Free base phthalocyanine, and aluminum, cadmium, magnesium, silicon, tin, and zinc metallated phalocyanines are reported to be fluorescent; see The Phthalocyanines 1:127, 1983. One or more of these species have been utilized in or proposed for semiconductors, organic dyes, stain removing agents, bactericides, and optical coatings. For example, European patent publication No. 142,369 discloses the use of certain phthalocyanine derivatives for hematology, specifically to differentiate basophils from other blood cells. U.S. Pat. No. 4,816,386 discloses a near-infrared-sensitive phthalocyanine-polymer composition comprising a substituted aluminum phthalocyanine and a polymer wherein substituted aluminum phthalocyanine dimers and/or dimer aggregates, which are reportedly responsible for the near-infrared sensitivity, are included.
Phthalocyanines have been reported for potential use in various types of immunoassays. See: U.S. Pat. No. 4,160,645 (at column 18, lines 18 to 22); U.S. Pat. No. 4,193,983 (at column 16, lines 36 to 39); U.S. Pat. No. 4,220,450 (at column 17, lines 23 to 26); U.S. Pat. No. 4,233,402 (at column 24, lines 53 to 56); U.S. Pat. No. 4,235,869 (at column 11, line 67 to column 12, line 2); U.S. Pat. No. 4,256,834 (at column 21, lines 34 to 36); U.S. Pat. No. 4,277,437 (at column 17, lines 11 to 14); U.S. Pat. No. 4,318,707 (at column 9, lines 14 to 16); U.S. Pat. No. 4,483,929 (at column 6, lines 36 to 39); U.S. Pat. No. 4,540,660; U.S. Pat. No. 4,540,670 (at column 11, lines 40 to 59); U.S. Pat. No. 4,560,534 (at column 5, line 67 to column 6, line 7); U.S. Pat. No. 4,650,770 (at column 18, lines 22 to 25); U.S. Pat. No. 4,656,129; and European patent publication No. 63,852 A3 (at page 31, lines 30 to 33). However, no mention is made of reactive forms of the phthalocyanine molecule which can be covalently coupled to a member of a ligand-binding partner.
Phthalocyanine derivatives have been employed as catalysts in chemiluminescence immunoassay systems. See: Hara, T., et al., Bull. Chem. Soc. Jpn. 56:2965-2968, 1983; Hara, T., et al., Bull. Chem. Soc. Jpn. 56:2267-2271, 1983; Hara, T., et al., Bull. Chem. Soc. Jpn. 57:587-588, 1984; Hara, T., et al., Bull. Chem. Soc. Jpn. 57:3009-3010, 1984; and Hara, T., et al., Bull. Chem. Soc. Jpn. 58:1299-1303, 1985. Hara described a chemiluminescence complex catalyst immunoassay in which iron phthalocyanine serves as the catalyst for a chemiluminescent reaction between luminol and hydrogen peroxide. The chemiluminescent signal is quantitated and correlated with the amount of analyte present in the test sample. Hara examined a number of phthalocyanine (Fe, Co) and porphyrin (Fe, Pd, Pt, Mn, Sn) complexes and reported that iron phthalocyanine exhibits the greatest catalytic activity and provides the highest sensitivities for this type of assay. Although Hara et al. describes a method for the covalent coupling of porphyrin and phthalocyanine complexes to proteins, the resultant product is highly aggregated. From spectral data contained in Bull. Chem. Soc. Jpn. 56:2965-2968, it is possible to calculate that the most monomerically labeled protein in this work contains less than 10% phthalocyanine monomer.
Phthalocyanines have also been suggested for use in photodynamic therapy (PDT), which is a radiation therapy for cancer that utilizes a photosensitive agent (sensitizer) and visible light as the radiation source. The sensitizer must be selectively delivered to the tumor tissues; for example, monoclonal antibody-hematoporphyrin conjugates have been reported. See: Mew, D., et al., J. Immunol. 130(3):1473-1477, 1983; and Mew, D., et al., Cancer Research 45:4380-4386, 1985. Thereafter, activation of the sensitizer by visible light kills the cells by a photodynamic reaction involving singlet oxygen generation. The phthalocyanines, particularly the aluminum and zinc tetrasulfonate derivatives, have been suggested for use in PDT, based upon their use as photosensitizers for cultured mammalian cells. See: Ben-Hur, E., and I. Rosenthal, Int. J. Radiat. Biol. 47:145-147, 1985; Ben-Hur, E., and I. Rosenthal, Photochem. and Photobiol. 42:129-133, 1985; Ben-Hur, E., and I. Rosenthal, Radiat. Res. 103:403-409, 1985, Brasseur, N., et al., Photochem. and Photobiol. 42:515-521, 1985; Ben-Hur, E., and I. Rosenthal, Lasers in the Life Sciences 1:79-86, 1986; Ben-Hur, E., I. Rosenthal, Photochem. and Photobiol. 43:615-621, 1986; Chan, W. S., et al., Br. J. Cancer 53:255-263, 1986; Rosenthal, I., et al., Radiat. Res. 107:136-142, 1986; Selman, S. H., et al., J. Urology 136:141-145, 1986; Ben-Hur, E., et al., Int. J. Radiat Biol. 51:467-476, 1987; Jori, G., et al., in Porphyrin Photosensitization, D. Kessel and T. J. Dougherty, eds., Plenum Press, New York, pp. 193-212, 1981; Spikes, J. D., and J. C. Bommer, Int. J. Radiat. Biol. 50(1):41-45, 1986; Spikes, J. D., Photochem. and Photobiol. 43(6):691-699, 1986; Langlois, R., et al., Photochem. and Photobiol. 44(2):117-123, 1986; Ben-Hur, E., et al., Photochem. and Photobiol. 46(5):651-656, 1987; Brasseur, N., et al., Photochem. and PhotobioI. 46(5):739-744, 1987; Singer, C. R. J., et al., Photochem. and Photobiol. 46(5):745-749, 1987; Tralau, C. J., et al., Photochem. and Photobiol. 46(5):777-781, 1987; Chan, W.-S., et al., Photochem. and Photobiol. 46(5):867-871, 1987; and Rosenthal, I., et al., Photochem. and Photobiol. 46(6):959-963, 1987. Of these, the following are considered to be the most pertinent.
Jori et al. (1981) address factors governing porphyrin sensitized photooxidations in various media. The efficiency of photooxidation was determined to be dependent upon the composition of the solvent in which the oxidation occurs.
Spikes and Bommer (1986) describe the photoproperties of zinc tetrasulfophthalocyanine in aqueous media. In water, the zinc derivative is aggregated and is incapable of photosensitization. In the presence of a cationic detergent, the zinc derivative disaggregates and becomes an efficient photosensitizer.
Langlois et al. (1986) observe the monomeric nature of sulfonated phthalocyanines of aluminum and gallium in water. Both were found to be efficient photosensitizers.
Pursuant to the present disclosure, while some metallo sulfonated phthalocyanines may be monomeric in water, it is not possible to covalently couple them to carriers such as proteins or oligonucleotides. The reactive form of the preferred sulfonated phthalocyanines are not soluble or monomeric in water. Monomerism of the reactive form of the phthalocyanine prior to and during covalent coupling is absolutely necessary to produce conjugates which bear monomerically tethered phthalocyanines.
Other fluorescent compounds (fluorophores) have been widely used in immunoassays, flow cytometry, and fluorescence microscopy. U.S. Pat. No. 4,614,723 is of interest for disclosing water-soluble porphyrin derivatives as label molecules for fluorescence immunoassays. The coupling of the disclosed porphyrin derivatives to immunologically active materials is reportedly carried out in the customary manner, e.g., with a water-soluble carbodiimide derivative.
It is also noteworthy that the most sensitive enzymatic immunoassays employ fluorogenic rather than colorimetric substrates. Three well-known fluorogenic enzyme substrate couples are: alkaline phosphatase (AP) and 4-methylumbelliferylphosphate (MUP); .beta.-galactosidase (.beta.-Gal) and 4-methylumbelliferyl-D-galactopyranoside (MUG); and horseradish peroxidase (HRP) and p-hydroxyphenyl acetic acid (HPA). Generally, the AP, .beta.-Gal, and HRP systems are useful for detection of analytes at concentrations greater than 10.sup.-15 M. To date, the sensitivity of these systems is limited by the spectral properties of the generated fluorophores.
Also of interest are prior publications concerning aggregation of phthalocyanines in solution, and the effects of solvents upon the absorption spectra of dyes generally. See: Gruen, L. C., Aust. J. Chem. 2,5:1661-1667, 1972; Blagrove, R.J., Aust. J. Chem. 26:1545-1549, 1973; Sheppard, S. E., and A. L. Geddes, J. Amer. Chem. Soc. 66(12):1995-2002, 1944; Sheppard, S. E., and A. L. Geddes, J. Amer. Chem. Soc. 66(12):2003-2009, 1944; Bernauer, K., and S. Fallab, Helv. Chim. Acta 44(5):1287-1292, 1961; and Darwent, J. R., et al., J. Chem. Soc., Faraday Trans. 2, 78:347-357, 1982. Of these, the following are considered to be the most pertinent.
Blagrove (1973) investigated the effect of urea and thiourea on the aggregation of copper phthalocyanine tetrasulfonic acid in water. Both were shown to disaggregate the dye in aqueous solution.
Gruen (1972) studied the visible absorbance spectra of two copper phthalocyanine dyes as a function of concentration, temperature, pH, ionic strength, and solvent composition. The data indicate an equilibrium between monomeric and dimeric dye exists. The equilibrium was most effected by the dielectric strength of the solvent systems studied, as the amount of monomer increased with decreasing dielectric constant.
Darwent et al . (1982) describe the photophysical properties of aluminum sulfophthalocyanine. No aggregation of the dye was observed in water over the concentration range studied.
Pursuant to the present disclosure, we have determined that the reactive form of aluminum phthalocyanine required for covalent coupling is neither soluble nor monomeric in water. The use of urea, thiourea, or organic solvents alone are insufficient for monomeric coupling. The reactive dye is optimally coupled to biological molecules as disclosed below in Examples 3 and 4. The composition of the reaction mixture including ingredients and concentration as well as timing and temperature are critical for achieving monomeric conjugation.